In nature, bacteria grow predominantly within sessile, matrix-enclosed communities known as biofilms, rather than as unattached planktonic cells. Biofilms protect resident bacteria and complicate many chronic infections by preventing immune function, compromising antimicrobial therapy, and dispersing planktonic cells that spread infection to distant body sites. Our long-term goal is to obtain fundamental understanding of the interrelated structural, enzymatic, and regulatory elements required for biofilm formation and dispersal as a prerequisite for developing approaches to combat biofilm-related infections. While diverse structural components and regulatory stragtegies affect biofilm formation, we hypothesize that there are a few critical factors that are of importance in many species, which are best studied in model organisms. Furthermore, one such factor is the RNA-binding protein CsrA (RsmA), a global regulator that controls biofilm formation in many species. In Escherichia coli, CsrA represses biofilm formation, while it activates biofilm dispersal and motility. The most important role of CsrA in biofilm formation is to inhibit translation and stimulate decay of pgaABCD mRNA, which is needed for the production and transport of poly-beta-1,6-N-acetyl-D-glucosamine (PGA). This polysaccharide adhesin stabilizes biofilms of diverse species and promotes disease transmission and/or virulence in certain pathogens. Regulators of pgaABCD gene expression have profound effects on biofilm development, incuding NhaR, a transcriptional activator, and CsrD, a novel Csr-system component. Our preliminary studies reveal two additional important regulatory systems that control biofilm formation and PGA production without affecting pgaABCD gene expression. In the next phase of this project, we propose to: 1) Elucidate two novel regulatory mechanisms of PGA polysaccharide production and biofilm formation. We will use in vivo and vitro polysaccharide synthesis and gene expression assays, transposon mutagenesis, and other molecular genetic approaches to accomplish this aim. 2) Delineate the functions of the pga genes and PGA itself. Effects of nonpolar deletions and site-directed mutations on polysaccharide polymerization, modification, localization, and chemical properties will be studied by biochemical and microscopic approaches. Properties of the PGA polysaccharide that determine its role as an adhesin will be examined. 3) The molecular genetic and biochemical mechanisms underlying biofilm dispersal will be examined. We will systematically determine the effects of CsrA induction (in preformed biofilm) on PGA levels, structure, and localization during the dispersal process. In the unlikely case that PGA is not involved in dispersal, we will examine possible roles of other envelope components. PUBLIC HEALTH RELEVANCE: Bacterial biofilms are surface-associated, matrix-enclosed microbial communities that are prevalent throughout the biosphere. In a variety of infections, biofilm formation protects the bacteria against the immune system and complicates antibiotic therapies. Biofilm-based infections are noted for causing disease in conjunction with the use of catheters, prosthetics and other devices. The long-term goal of this study is to obtain fundamental information about the interrelated structural, enzymatic and regulatory elements that are required for biofilm formation and dispersal. The resulting information will bolster efforts to develop new strategies and therapeutic approaches to combat biofilm-related infections.